CN111249466B - Application of Tanshinone IIA combined with miR-29b inhibitor in the preparation of drugs for the treatment of tendon adhesions - Google Patents

Abstract

The invention relates to the application of a combination of tanshinone IIA and a miR-29b inhibitor in the preparation of a medicament for treating tendon adhesion, and belongs to the technical field of medicine. The experimental results of the present invention show that the use of miR-29b inhibitors on tendon stumps can activate the TGF-β1/Smad3 pathway and initiate the endogenous repair mechanism, and subsequent use of TSA can block the TGF-β1/Smad3 pathway and inhibit exogenous repair mechanism. Therefore, the combination of the two treatments can prevent tendon adhesions and ensure tendon strength. The combination of tanshinone IIA and miR-29b inhibitor can prepare a drug for the treatment of tendon adhesion, which brings new ideas for clinical prevention and treatment of tendon adhesion.

Description

Application of Tanshinone IIA combined with miR-29b inhibitor in the preparation of drugs for the treatment of tendon adhesions

technical field

The invention relates to the application of a combination of tanshinone IIA and a miR-29b inhibitor in the preparation of a medicament for treating tendon adhesion, and belongs to the technical field of medicine.

Background technique

Tendon adhesion refers to the dysfunction of tendon movement caused by the proliferation and invasion of surrounding tissues during the repair of tendon injury. Post-traumatic tendon adhesion is one of the common problems after tendon injury. Due to the prevalence of adhesion and the contradictory factors that tendon healing requires sufficient strength to perform early functional exercise, there is still no effective solution so far. Tendon adhesions can occur due to mechanical and chemical injuries. After injury, interstitial cells and mast cells are induced to release a variety of active substances, including thrombin, histamine, heparin and other vasoactive substances, causing tissue inflammatory response, increased capillary permeability, and a large amount of serous exudation. If these exudates cannot be decomposed and absorbed, fibrin will accumulate, followed by infiltration of fibroblasts and ingrowth of new capillaries, eventually forming adhesions. From the perspective of tendon healing, it is currently believed that tendon healing is accomplished by both internal and external repair mechanisms. The endogenous repair mechanism repairs the damaged tendon through tenocyte proliferation, while the exogenous repair mechanism repairs the damaged tendon through the proliferation of peritendon tissue, fibroblasts and inflammatory cells, so the strength of the tendon itself mainly depends on the endogenous repair mechanism. , while exogenous repair mainly forms adhesions. Therefore, the ideal tendon repair mode is to start the endogenous mechanism in advance at the stump of the tendon, so that the tendon has sufficient strength for healing, and then prevent the adhesion by inhibiting the exogenous mechanism in the peri-tendon tissue.

The main cytokines found to be related to the repair process of tendon injury include: transforming growth factor-β (TGF-β), bone morphogenetic protein-12 (BMP-12), insulin-like growth factor 1 (insulin-like growth factor-Ⅰ, IGF-1), basic fibroblast growth factor (basic fibroblast growth factor, bFGF), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), platelet-derived growth factor (plateledderived growth factor) growth factor, PDGF) and so on. Among them, TGF-β1/Smad3 is a common important signaling pathway in the process of wound repair, and it is one of the factors of pathological fibrosis in tissues. Park Yingjie et al. observed the dose effect and time effect of TGF-β1 on human embryonic tenocytes and the effect of TGF-β1 on cell cycle and proliferation index, and found that TGF-β1 can stimulate tenocyte DNA synthesis, and pointed out that TGF-β1 It may be involved in tendon repair by promoting tenocyte proliferation. Through the study of sheep embryonic tendon injury model, Beredjiklian et al. found that the embryonic tendon healed without scarring, while the content of TGF-β1 and its mRNA in the embryonic environment was significantly lower than that in the adult tissue, suggesting that the scarless healing mechanism of embryos may be this kind of caused by low concentrations of TGF-β1. Malmstrom et al. found that TGF-β1 acts by promoting the recruitment of fibroblasts and macrophages, angiogenesis, stimulating collagen production, downregulating the activity of matrix metalloproteinases and increasing the activity of matrix metalloproteinase inhibitors. A cytokine that plays an important role in acute inflammation and wound healing, its overexpression can lead to excessive scarring and fibrosis.

Tanshinone IIA is the active ingredient of Salviamiltiorrhiza bunge, a plant of the Lamiaceae family. Liu Chenghai et al. stimulated the activation of HSCs in primary cultured rats in vitro stimulated by TGF-β1, and at the same time acted on salvianolic acid, and found that salvianolic acid inhibited cell collagen secretion and α-smooth muscle actin (α-smooth muscle actin, α-SMA) Compared with the protein expression of plasminogen activator inhibitor, and inhibited the protein expression of Smad 2 and 3 in the cytoplasm and nucleus, it indicated that the mechanism of salvianolic acid against liver fibrosis and inhibition of HSC activation lies in inhibiting the signal transduction of TGFβ1 in HSC. In the liver fibrosis experiments in mice, it was also found that tanshinone IIA promoted the expression of collagen type I mRNA and decreased the expression of IGFBP7 in liver tissue by inhibiting the TGF-β1/Smad3 signaling pathway, thereby exerting its anti-hepatic fibrosis effect. Sun Xingwang et al. confirmed that tanshinone IIA reduced renal fibrosis in 5/6 nephrectomy rats by regulating TGF-β1/Smad and NF-κB pathways. Meanwhile, sodium tanshinone IIA sulfonate ameliorated bladder fibrosis in a rat model of partial bladder outlet obstruction by inhibiting TGF-β/Smad pathway activation.

Chinese medicine is an important part of the splendid culture of the Chinese nation, and it is also one of the most influential disciplines in the world. The concept of "preventing disease" in traditional Chinese medicine has a long history. In the first chapter of "The Great Theory of Four Qi Regulations" in "The Yellow Emperor's Classic of Internal Medicine", "the sage cannot cure the disease and cure the disease, and if it is not cured, the disorder will cure the disorder.... It is not too late to take medicine after it has been completed, and to cure it after chaos has been completed, such as piercing a well when thirsty, or casting a cone when fighting. The theory of "stasis at the beginning of arthralgia" guides the treatment of early tendon adhesions, which belongs to the aspect of "preventing disease and preventing changes". Consistent with the strategy of "prevention-to-treatment". The use of drugs to prevent pre-disease or high-risk groups has naturally become a hot spot in international evidence-based medicine. This is a trend in the global healthcare industry. Therefore, the intervention of early tendon adhesion is the needs of patients and the development of health services.

Tendon adhesion is a common complication after tendon injury, and its prevention and treatment has always been a common concern in the scientific community. How to make the tendon less or no adhesion after injury or repair, and restore its sliding function as soon as possible without affecting the healing of the tendon itself is an existing technical problem, and there is still no effective drug and method to completely solve the problem of tendon adhesion. question. Local injection of traditional Chinese medicine has the advantages of overall dynamic adjustment and individualized treatment. At the same time, the precise and rapid treatment effect of western medicine is also the guarantee of good patient compliance. Therefore, the use of traditional Chinese medicine combined with gene therapy for early prevention of tendon adhesion is urgently needed in clinical practice. Conform to the needs of the country, society and individuals, bring tangible benefits to patients, and bring great economic and social benefits to the country, society, families and individuals.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a combination of tanshinone IIA and a miR-29b inhibitor in the preparation of a drug for treating tendon adhesion, so as to solve the problems in the prior art.

The technical scheme adopted by the present invention to solve its technical problems is:

The application of a combination of tanshinone IIA and a miR-29b inhibitor in the preparation of a medicament for treating tendon adhesions.

Preferably, said application is the use of Tanshinone IIA after a miR-29b inhibitor.

Preferably, the application is the use of Tanshinone IIA 0-72 h after the miR-29b inhibitor. Optimally, the described application is tanshinone IIA 4-8 h after miR-29b inhibitor.

A drug for the treatment of tendon adhesion, which contains tanshinone IIA and a miR-29b inhibitor. Optimally, the drug is administered by injection.

miRNA is a short-chain non-coding RNA that participates in the translational repression of genes and the degradation of mRNA, and plays an important role in metabolism, cell proliferation, differentiation, apoptosis and development. A study of patients with liver fibrosis found that serum levels of miRNA-16, miRNA-146a, miRNA-221 and miRNA-222 were significantly up-regulated in both early and late stages of liver fibrosis. Liu et al. found that miR-21 was significantly up-regulated in the lungs of bleomycin-induced fibrotic mice and the lungs of IPF patients, and the increased levels of miR-21 promoted the promotion of TGF-β1 in fibroblasts. fibrosis, leading to the development of pulmonary fibrosis. Decreased levels of miRNA-29 were also found to be associated with fibrosis in multiple organs including heart, liver, kidney and skin, as well as SSc, suggesting that it may be a core "fibromiRNA". After knockdown of miR-29 in human fetal lung fibroblast IMR-90 cells, it was found that the expression of many fibrosis-related genes upregulated by TGF-β1 was significantly reduced, and collagen was inhibited, confirming the effect of miR-29 on the process of lung fibrosis. The influence of the important pathway TGFβ1. In diabetic rats, miR-29b can inhibit the occurrence of diabetic nephropathy by negatively regulating TGF-β1/Smad3-mediated fibrosis and inhibiting Sp1/NF-κB-driven inflammatory responses. In the study of rat Achilles tendon cells, our group found that miR-29b inhibitor could reverse the changes of miR-29b, TGF-β1, Smad3 and P21 expression caused by chitosan.

Considering that the regulatory effect of tanshinone IIA and miR-29b on tissue fibrosis can be achieved through the TGF-β1/Smad3 pathway, the invention innovatively adopts the combination of tanshinone IIA and miR-29b inhibitor, that is, combining traditional Chinese medicine and gene A treatment method is provided to realize the clinical treatment of tendon adhesion, and to prepare medicines for the treatment of tendon adhesion.

The invention verifies the inhibitory effect of tanshinone IIA (TSA) combined with miR-29b inhibitor on the formation of adhesions after tendon injury through molecular cells and biological multi-level. All data are presented as mean ± standard deviation. ANOVA was used to compare three or more groups, and Student's t-test was used to compare between two groups. All analyses were done using SPSS 17.0 software. P<0.05 was considered to be statistically significant.

In the present invention, the optimal rat in vivo application concentration of TSA is firstly determined to be 0.1 μM through in vitro experiments, and negative control, TSA, miR-29b interference adenovirus, and TSA and miR-29b interference adenovirus are carried out in primary rat fibroblasts. Intervention to observe cell proliferation and protein expression. The results showed that there were statistical differences between the treatment group and the control group and between the treatment groups (P<0.05). In terms of the expression of inflammatory factors, TSA treatment decreased the expression of TGF-β1 and Samd3 levels, and miR-29 inhibitor significantly up-regulated the expression of TGF-β1 and Smad3, and the combined application of the two significantly increased the expression levels of TGF-β1 and Samd3 higher than in cells treated with TSA alone, but significantly attenuated compared with cells treated with miR-29b inhibitor. This suggests that both TSA and miR-29b inhibitors target the same pathway, implying that the combination of the two can trigger the endogenous pathway and manipulate the late-stage targeting of the exogenous pathway. CCK-8 analysis and fluorescence-activated cell sorting (FACS) analysis showed that cells treated with miR-29b inhibitor significantly increased cell proliferation, while TSA-treated cells significantly decreased cell proliferation, when TSA and miR-29b inhibitor When acting on cells simultaneously, the cell proliferation ability was significantly lower than that of miR-29b inhibitor, but higher than that of TSA-treated cells, and the same trend was observed in the apoptosis analysis.

Further, in the in vivo experimental study carried out by the present invention, all rats were randomly divided into 6 groups, 1 group was a sham operation group without tendon injury treatment, and the remaining 5 groups of rats were treated with Achilles tendon injury after making a rat model of Achilles tendon injury. The markers were located at the tendon injury, and they were randomly divided into 4 groups of miR-29b inhibition group (treatment group) and 1 group of negative control injection group (control group). Transfection injection. The four groups in the treatment group were injected with 0.5 ml of tanshinone IIA injection at the Achilles tendon mark immediately after operation, 6 hours after operation, 24 hours after operation, and 72 hours after operation, and each injection lasted for 1 week. Rats were observed for general behavior and sacrificed after 3 weeks. The tissue from the injured area of Achilles tendon was taken for gross observation, histopathological analysis and biomechanical measurement. Cell-level results showed that combined treatment with TSA and miR-29b inhibitor significantly reduced the number of apoptotic cells, and the level of apoptotic cells in the miR-29b inhibitor combined with TSA injection 6 hours after surgery was the closest to the untreated group. At the molecular level, the levels of TFG-β and Smad3 in the treatment group were significantly lower than those in the control group, but still higher than those in the untreated group; at the same time, compared with the control group, the levels of collagen type I and cyclin D were significantly increased in the treatment group, but not in the control group. There was no significant difference within the group. In histological observation, in the group treated with miR-29b inhibitor and TSA, the number of collagen fibers and fibroblasts increased and arranged well, while the number of collagen fibers and fibroblasts in the control group increased significantly and the arrangement was irregular. The general study showed that the Achilles tendon adhesion in the treatment group was less than that in the control group, and the maximum load of the tendon under all treatment conditions was higher than that in the control group, P<0.05, there was a statistical difference. And the maximum load of the tendon in all treatment conditions was higher than that in the control group.

The experimental results of the present invention show that the use of miR-29b inhibitor at the tendon stump can activate the TGF-β1/Smad3 pathway and initiate the endogenous repair mechanism, and then the use of TSA can block the TGF-β1/Smad3 pathway and inhibit the exogenous repair. mechanism. Therefore, the combination of the two treatments can prevent tendon adhesions and ensure tendon strength. The combination of tanshinone IIA and miR-29b inhibitor can prepare a drug for the treatment of tendon adhesion, which brings new ideas for clinical prevention and treatment of tendon adhesion.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the following will briefly introduce the accompanying drawings used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only For some embodiments of the present invention, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

Figure 1 shows the dynamic changes of miR-29b, TGF-β1 and Smad under the action of miR-29b interfering with adenovirus and TSA, in which, A: MTT assay to measure the cytotoxicity of TSA, B: qPCR to detect the expression of miR-29, C : TGF-β1 mRNA and protein expression levels were measured under different conditions, D: Smad mRNA expression was detected by qPCR (n=3) under different conditions, and protein expression levels were detected by western blotting (n=1);

Figure 2 shows the effect of miR-29b interfering with adenovirus and TSA treatment on cell proliferation, apoptosis and cell cycle, A: cell proliferation detected by CCK8 kit, B: cell apoptosis detected by FACS, C: primary isolation under different conditions Cell cycle analysis of cells;

Figure 3 is a graph showing the effect of drugs on the expression of miR-29b and apoptosis after the establishment of a rat Achilles tendon injury model, in which, A: qPCR quantifies the expression of miR-29b, B: TUNEL method detects the frequency of apoptotic cells;

Figure 4 shows the effect of miR-29b inhibitor and TSA treatment on the expression of TGF-β1, p21 and Smad3, A: TGF-β1 mRNA expression detected by qPCR method, n=3; B: p21 mRNA expression detected by qPCR method, n=3 ;C: qPCR detection of Samd3 mRNA expression;

Figure 5 is the analysis diagram of collagen expression and histological changes in tendon tissue, wherein, A: the production of type I collagen in each group, B: the production of type III collagen in each group of rat tendon tissue, C: hematoxylin and eosin staining (HE staining) and Masson staining (x200), D: expression of cyclin D;

Figure 6 is a diagram of tendon strength analysis, wherein, A: biomechanical analysis of maximum load, B: evaluation of periintimal adhesions;

In Figures 2 to 6, n=3, p-values *<0.05, **<0.01, ***<0.005, ****<0.001.

Detailed ways

The technical solutions of the present invention will be further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following examples, and any modifications and/or changes made to the present invention will fall within the protection scope of the present invention.

In the present invention, unless otherwise specified, all parts and percentages are in units of weight, and the equipment and raw materials used can be purchased from the market or commonly used in the art. The methods in the following examples, unless otherwise specified, are conventional methods in the art.

The reagents used in the following examples, unless otherwise specified, can be purchased from conventional biochemical reagent stores.

In the following examples: miR-29b, AAV-293 cells, miR-29b interfering adenovirus, and miR-29b inhibitor were all provided by Guangzhou Ribo Biotechnology Co., Ltd.

The core of the present invention is to provide a combination of tanshinone IIA and a miR-29b inhibitor in the preparation of a drug for treating tendon adhesions, which is referred to as specific embodiment 1. The method includes the following steps:

Example 1

1. miR-29b interferes with adenovirus customization

The miR-29b interfering recombinant plasmid was constructed using the miRNA sponge structure, and co-transfected with the packaging plasmids pHelper and pAAV-RC into AAV-293 cells; 3 days after transfection, the cells were lysed to collect AAV virus particles; centrifugation and ultrafiltration were used to concentrate and purify the virus The supernatant; the virus titer was determined by fluorescence quantitative PCR, and the total amount of miR-29b interfering adenovirus (miR-29bshRNA adenovirus) and empty adenovirus (miR-29b NC adenovirus) was 1^10^ each. 11pfu.

2. Creation of rat Achilles tendon injury model

Experimental animals: SD rats, male, about 350 g, SPF grade, provided by the Experimental Animal Center of Zhejiang University School of Medicine.

Rat Achilles tendon injury model: intraperitoneal injection of halothane (50mg/kg). Tourniquet on the upper right hind limb, longitudinal incision of the heel, opening of the Achilles tendon sheath, picking out the Achilles tendon and cutting it horizontally, using 5-0Ti-Cron non-absorbable suture (COVIDIEN, Langhorne, PA, USA); Tendon sheaths and skin closures were sutured directly with 5-0 Surgipro sutures (COVIDIEN, Langhorne, PA, USA). After the model was established, a line or mark was placed on the Achilles tendon injury to facilitate the positioning of the drug, and the rat hindlimb was splinted.

3. Isolation and culture of primary rat fibroblasts

The tendon at the injury site of the SD rat Achilles tendon after modeling was taken, rinsed three times with ampicillin-streptomycin sulfate-PBS, and then the peritendon tissue was removed. 200U/ml collagenase IV in DMEM medium, digested at 37°C for 2 hours. Centrifuge at 1200 rpm for 10 minutes, wash the pellet with serum-free DMEM, centrifuge twice, resuspend in DMEM medium containing 10% fetal bovine serum, add it to a petri dish, and adhere to the wall for 2 hours at 37°C, 5% CO ₂ . After removing the original medium, the fibroblasts were isolated and routinely cultured and passaged. The isolated cells were identified as fibroblasts by conventional HE staining microscopy and immunocytochemical staining to detect the expression of Col1 and Col3.

Example 2 Effects of TSA and miR-29b inhibitors on rat fibroblasts

1. MTT (monocyte direct cytotoxicity assay) screening the most suitable tanshinone concentration

The rat fibroblasts in logarithmic growth phase and in good growth state in Example 1 were digested and made into a single-cell suspension, inoculated in a 96-well plate at a density of 2000/well, and the culture medium was added to the final volume. 100 μl, set up 1 blank control group, 6 different concentrations of TSA treatment groups (0.001uM, 0.01uM, 0.1uM, 1uM, 10uM, 20uM), each group set 5 duplicate wells, at 37 ° C, 5% CO ₂ The cells were cultured under the environment, and the cells were cultured for 24 hours after adherence, and 20 μl of 5 mg/ml MTT was added for 4 hours. The MTT-containing medium was discarded, and 150 μl of DMSO was added to incubate for another 30 min, and the absorbance at 490 nm was detected with a multi-function microplate reader. The absorbance value reflects the cell viability. The test results are shown in Figure 1A, the results show that 1 μM TSA significantly reduces the cell viability, therefore, the inventors used 0.1 μM TSA in this study.

In the following test, the rat fibroblasts in the logarithmic growth phase and in good growth state in Example 1 were digested and made into a single-cell suspension, which was inoculated in a 96-well plate at a density of 2,000 cells/well, and the culture medium was added to The final volume was 100 μl, and two negative control groups were set up, one blank control group and one miR-29b NC adenovirus transfection group; and four treatment groups, which were miR-29b interfering adenovirus transfection group, 0.1 μM TSA treatment group, miR-29b NC adenovirus + 0.1 μM TSA treatment group, miR-29b interfering adenovirus + 0.1 μM TSA treatment group, each group was set up with 5 duplicate wells, at 37 °C, 5% CO ₂ environment The cells were cultured for 24 h after the cells adhered.

2. Detection of cell proliferation by CCK8: trypsin digestion, collect cells in logarithmic growth phase, resuspend cells at a density of 1 × 105 cells/ml, and plate them in 96-well plates. The absorbance value OD450 of each well was measured by standard instrument to detect the level of cell proliferation. The experimental results are shown in Figure 2A.

As can be seen from Figure 2A, compared with the negative control group, cells treated with miR-29b interfering with adenovirus significantly increased cell proliferation, while TSA-treated cells significantly decreased cell proliferation, and when TSA and miR-29b inhibitors acted simultaneously on cells, the inventors found that compared with miR-29b inhibitor, the cell proliferation ability was significantly reduced and higher than that of TSA-treated cells.

3. Detection of cell cycle by flow cytometry: cells were incubated with different concentrations of TSA for 24 hours, then stained with propidium iodide, and DNA content was detected by flow cytometry.

The cells of each group in the logarithmic growth phase were taken, and then fixed and permeabilized in pre-chilled 100% methanol. Propidium iodide (50 μg/ml) and RNase A (125 μg/ml) were added at room temperature, and incubated for 45 minutes for staining. The cells were filtered with 200-mesh nylon omentum, and the cell cycle was analyzed using a flow cytometer FACSCalibur (Becton Dickinson). (See Figure 2C for the test results).

The isolated and cultured fibroblasts were collected, digested with trypsin, washed with pre-cooled PBS, and resuspended in 100 μl binding buffer. Then 2 μl Annexin V-FITC and 5 μl PI were added to the cells and incubated for 15 minutes at room temperature in the dark. Add 400 μl of binding buffer and then react on the machine. The software analyzes the apoptosis rate (see Figure 2B for the test results).

The above experimental results showed that the proportion of cells in the G1 phase was higher in the TSA-treated cells, and the apoptosis rate induced by TSA was higher than that in the control cells. In addition, compared with the miR-29b-interfering adenovirus-treated group, the proportion of cells in G2 and S phases in the TSA-treated group was decreased. At the same time, when TSA and miR-29b interfering adenovirus acted on cells at the same time, the inventors also observed that apoptosis was significantly reduced, and the proportion of cells in G1 phase was slightly higher than that of pure TSA, while the opposite was true for miR-29b inhibitors.

Taken together, the results suggest that miR-29b can promote cell proliferation and TSA can reduce these effects.

4. Changes in the content of TGF-β1, Smad3, and miR-29b detected by QPCR: take the cells of each group in the logarithmic growth phase, digest with 0.25% trypsin, centrifuge the solution at 1200 rpm for 10 minutes, wash with PBS, and centrifuge the cells twice by Trizol method Total RNA was extracted, and the concentration of total RNA was determined by UV spectrophotometry. The primer sequences described in Table 1 (the same below) and real-time PCR kit (TaKaRa) were used to detect TGF-β1, Smad3, and miR-29b, and 2-△△ The relative ratio of gene expression was calculated by CT method, and U6 was used as the internal reference gene. The test results are shown in Figure 1B, C (left), and D (left).

Table 1 Primers used in QPCR detection

Universal primer for mircoRNA antisense strand: R primer: CCAGTGCAGGGGTCCGAGGTATT (SEQ ID No. 11).

From the data in Figure 1B, the inventors observed that the application of miR-29b to interfere with adenovirus significantly reduced the expression of miR-29b in fibroblasts, and TSA treatment significantly enhanced the expression of miR-29b, and the use of TSA and miR-29b to interfere with adenovirus simultaneously Treatment of cells counteracted the effect of treatment, indicating no significant changes in miR-29b in double-treated samples. From the data in Figure 1C and D, it can be seen that the dynamic changes of TGF-β1 and Smad expression at the mRNA and protein levels are the same.

5. Western Blot (WB) detection of TGF-β1, p-Smad3, Smad3: routinely extract the whole protein of the cells of each treatment group, centrifuge at 12000rpm, 4 ℃ for 15min, take the supernatant and use the BCA kit to measure the concentration of the extracted protein. 30 μg of total protein from each sample was separated by 10% SDS-PAGE gel electrophoresis. The wet protein was transferred to a 0.22-micron pore PVDF membrane. The membrane was incubated with the corresponding primary antibody overnight at 4°C, and then mixed with HRP-labeled secondary antibody. (1:2000) and incubated at room temperature for 90 min. Ez-ECL chemiluminescence detection kit was used for development, and the contents of p21, TGF-β1, Smad3 and p-Smad3 in the tissue were detected. Band grayscale analysis was performed by Quantity One software. β-actin was used as an internal reference protein. The test results are shown in Figure 1C (right) and D (right).

From the data in Figure 1C (right) and D (right), the inventors found that TSA treatment reduced the expression of TGF-β1 and Samd3 levels. In contrast, the miR-29 interfering adenovirus group significantly upregulated the expression of TGF-β1 and Smad3. When cells were treated with both TSA and miR-29 interfering adenovirus, the expression levels of TGF-β1 and Samd3 were significantly higher than in cells treated with TSA alone, but significantly attenuated compared with cells treated with miR-29b interfering adenovirus.

This result shows that both TSA and miR-29b inhibitors target the same pathway, which means that the combination of the two can trigger the endogenous pathway and manipulate the targeting late stage of the exogenous pathway.

Example 3 The preventive and therapeutic effects of TSA and miR-29b inhibitors on rat Achilles tendon adhesions

45 SD rats after modeling in Example 1 were randomly divided into 6 groups with 5 rats in each group. Group 1 was a sham operation group without tendon injury treatment, and the other 5 groups of rats were randomly divided into 4 groups for miR- The 29b inhibition group (treatment group) and group 1 were the negative control injection group (control group), and the miR-29b interfering adenovirus or empty virus was transfected and injected in vivo at the marked place every 3 days (10^9pfu/only). /Second-rate). The rats in the four groups of the treatment group were injected with 0.5 ml of TSA injection at the Achilles tendon mark immediately after operation, 6 hours after operation, 24 hours after operation, and 72 hours after operation, and each injection lasted for 1 week. They were killed after 3 weeks.

1. General observation: Dynamic observation of the rat's mental state, activity, fur, body weight, wound healing of the Achilles tendon, etc. was carried out. All the rats survived, the wounds healed in one stage, and there was no infection or disuse of the hind limbs.

2. General observation: After the SD rats were sacrificed, the skin and subcutaneous tissue of the operation area were incised, and the tissue at the suture of the tendon, the tendon sheath and the tendon were exposed respectively, and the inflammatory reaction, granulation hyperplasia, and tendon healing and adhesion of the tendon stump were observed. The scoring criteria are: grade I, no adhesion around the tendon; grade II, a small amount of limitation at the suture of the tendon, film-like adhesion, and slightly limited sliding of the tendon; Partially limited sliding; Grade IV, moderate dense adhesions in a large area, with a certain mobility, and tendon sliding is obviously limited; Grade V, dense adhesions in large areas, extensive adhesions between tendons and sheaths and subcutaneous, with extremely poor sliding. For tendon healing, grades I and II were excellent, grades III were good, and grades IV and V were poor. The test results are shown in Figure 6B, and the results show that the tendon healing in each treatment group is better than that in the control group.

3. Biomechanical measurement: After the rats were sacrificed, the Achilles tendon and the calcaneus connected by the Achilles tendon were dissected and separated, and the adhesion tissue around the tendon was cleaned up to prevent affecting the experimental results. Apply a preload of 1N, adjust the Achilles tendon tension and return to zero. The maximum distance was set at 6.50 mm, and the Achilles tendon was stretched to rupture at a speed of 0.05 mm/s (biomechanical slow-pull test) and 1 mm/s (biomechanical fast-pull test), respectively. Read the ultimate load and the actual tensile length of the Achilles tendon through the Wintest mechanical testing software, calculate its toughness = load/strain (N/mm), observe the fracture site, and record the original length of the tendon, strain ratio, maximum load, and maximum strain. . The test results are shown in Figure 6A.

As can be seen from the data in Figure 6A, the results showed that the maximum load under all treatment conditions was higher than that of the control group, although it was still lower than that of the untreated group, and the tendon could not be fully healed, but it still suggested that early induction of endogenous repair mechanisms could improve tendon strength. Reduce tendon adhesions.

4. Histological observation: The treated tendon tissue of each group of rats was immersed and fixed in 4% paraformaldehyde, decalcified, dehydrated, embedded in paraffin, and sliced for use. Hematoxylin and eosin staining (HE staining) and Masson's trichrome staining (x200) were used for histological observation and light microscope analysis: fibroblasts, collagen fibers, inflammatory cells. The test results are shown in Figure 5C. From the data in Figure 5C, in all groups, the inventors observed that compared with the untreated control group, the fibroblasts and collagen tissue at the repair site were proliferated and well arranged.

Figure 3 shows the establishment of a rat model, which was treated according to different conditions in groups 1 to 6 shown in Figure 3 . Rats were sacrificed after 3 weeks of treatment. The apoptosis rate was detected by TUNNEL staining, and the results under fluorescence microscope (Figure 3B) showed that the combined treatment of TSA and miR-29b inhibitor could significantly reduce the number of apoptotic cells.

Notably, the inventors observed slightly lower levels of apoptotic cells in the other treatment groups compared to rats treated with a miR-29b inhibitor first followed by TSA 6 hours later, but similar to the untreated group , indicating that different treatment time points can produce different therapeutic effects.

5. Molecular biological observation: QPCR detected the effect of miR-29b inhibitors in the rat model, and the results showed (Fig. 3A) that all inhibitor treatments significantly inhibited the expression of miR-29b RNA compared with the untreated group.

The expression levels of TGF-β1, p21 and Smad3 were detected by QPCR and WB. The results showed that (Fig. 4) TGF-β1, p21 and Smad3 were expressed at the same mRNA and protein levels. The treatment group significantly reduced their expression, but still higher than the healthy ones. control group, which suggests that the treatment may not fully heal the tendon and requires further detailed study.

The expression of collagen in tendon tissue was detected by immunohistochemistry. Figure 5A, B, and D are the analysis diagrams of collagen expression and histological changes in tendon tissue. The expression levels of collagen I in each group were detected by immunohistochemistry at 3 weeks. A: the production of type I collagen in each group, B: the production of type III collagen in the tendon tissue of the rats in each group, D: the expression of cyclin D: the histological evaluation of the tendon tissue in each group at the 3rd week. According to the results of Figure 5A, B and D, the expressions of collagen types I and III (Col I/III) in tendon tissues showed opposite trends; the protein level of Col I increased when TSA was added in a delayed manner, while cyclin D was significantly different from that without TSA. There was a significant increase compared to the treatment groups, but there was no difference between the treatment groups. The results confirmed that the combined application of TSA and miR-29b inhibitor could reduce collagen fiber expression and ultimately adhesion formation by inhibiting the TGF-β1/Smad3 pathway.

In vitro experiments showed that TSA treatment significantly enhanced the expression of miR-29b, TSA could reduce the expression of TGF-β and Smad3, inhibit cell proliferation and promote apoptosis, while miR-29b inhibitor did the opposite. In contrast, when TSA and miR-29 inhibitor were used together, apoptosis was significantly reduced, and the expression levels of TGF-β and Samd3 were significantly higher than those treated with TSA alone, but significantly higher than those treated with miR-29b inhibitor. weakened, proving that the combined application of miR-29b inhibitor and TSA can effectively prevent tendon adhesion and improve tendon strength. The mechanism of action is that miR-29b inhibitor can activate the TGF-β/Smad3 pathway, trigger the endogenous pathway, and induce high proliferation of fibroblasts. In vivo experiments showed that under all treatment conditions, the number and arrangement of collagen fibers and fibroblasts in rat Achilles tendon injury were better than those in the control group, and the maximum load of rat Achilles tendon was higher than that in disease models, especially in miR The addition of TSA after 6 h of -29b inhibitor treatment produced minimal cytotoxicity in our rat model and was more effective in inhibiting tendon adhesions and enhancing the strength of tendon adhesions after healing.

In conclusion, the present invention confirms that the use of miR-29b inhibitor on tendon stumps can initiate the endogenous repair mechanism, and the subsequent use of TSA can inhibit the exogenous repair mechanism, thereby achieving the purpose of reducing tendon adhesions. In particular, adding TSA after 6 hours of miR-29b inhibitor treatment produces less cytotoxicity in the rat model of the present invention, and has better effects on inhibiting tendon adhesion and enhancing the strength of tendon adhesion after healing.

The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same or similar parts between the various embodiments may be referred to each other. As for the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant part can be referred to the description of the method.

The application of the tanshinone IIA provided by the present invention in combination with the miR-29b inhibitor in the preparation of a medicament for treating tendon adhesions has been described in detail above. The principles and implementations of the present invention are described herein by using specific examples, and the descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

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Claims (5)

1. An application of tanshinone IIA and miR-29b inhibitor in preparation of a medicine for treating tendon adhesion is characterized in that: the application is that tanshinone IIA is used after a miR-29b inhibitor, and the miR-29b inhibitor is miR-29b interference adenovirus.

2. Use according to claim 1, characterized in that: the application is to use tanshinone IIA 0-72h after the miR-29b inhibitor.

3. Use according to claim 1, characterized in that: the application is to use tanshinone IIA 4-8h after the miR-29b inhibitor.

4. A medicament for treating tendon adhesion is characterized in that: the medicine comprises tanshinone IIA and a miR-29b inhibitor, wherein the miR-29b inhibitor is miR-29b interference adenovirus.

5. A medicament for the treatment of tendon adhesions as in claim 4, which is characterized by: the administration mode of the medicine is injection.

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